Molecular phylogeny of the family Rhabdiasidae (Nematoda: Rhabditida), with morphology, genetic characterization and mitochondrial genomes of Rhabdias kafunata and R. bufonis

Background The family Rhabdiasidae (Nematoda: Rhabditida) is a globally distributed group of nematode parasites, with over 110 species parasitic mainly in amphibians and reptiles. However, the systematic position of the family Rhabdiasidae in the order Rhabditida remains unsolved, and the evolutionary relationships among its genera are still unclear. Moreover, the present knowledge of the mitochondrial genomes of rhabdiasids remains limited. Methods Two rhabdiasid species: Rhabdias kafunata Sata, Takeuchi & Nakano, 2020 and R. bufonis (Schrank, 1788) collected from the Asiatic toad Bufo gargarizans Cantor (Amphibia: Anura) in China, were identified based on morphology (light and scanning electron microscopy) and molecular characterization (sequencing of the nuclear 28S and ITS regions and mitochondrial cox1 and 12S genes). The complete mitochondrial genomes of R. kafunata and R. bufonis were also sequenced and annotated for the first time. Moreover, phylogenetic analyses based on the amino acid sequences of 12 protein-coding genes (PCGs) of the mitochondrial genomes were performed to clarify the systematic position of the family Rhabdiasidae in the order Rhabditida using maximum likelihood (ML) and Bayesian inference (BI). The phylogenetic analyses based on the 28S + ITS sequences, were also inferred to assess the evolutionary relationships among the genera within Rhabdiasidae. Results The detailed morphology of the cephalic structures, vulva and eggs in R. kafunata and R. bufonis was revealed using scanning electron microscopy (SEM) for the first time. The characterization of 28S and ITS regions of R. kafunata was reported for the first time. The mitogenomes of R. kafunata and R. bufonis are 15,437 bp and 15,128 bp long, respectively, and both contain 36 genes, including 12 PCGs (missing atp8). Comparative mitogenomics revealed that the gene arrangement of R. kafunata and R. bufonis is different from all of the currently available mitogenomes of nematodes. Phylogenetic analyses based on the ITS + 28S data showed Neoentomelas and Kurilonema as sister lineages, and supported the monophyly of Entomelas, Pneumonema, Serpentirhabdias and Rhabdias. Mitochondrial phylogenomic results supported Rhabdiasidae as a member of the superfamily Rhabditoidea in the suborder Rhabditina, and its occurrance as sister to the family Rhabditidae. Conclusions The complete mitochondrial genome of R. kafunata and R. bufonis were reported for the first time, and two new gene arrangements of mitogenomes in Nematoda were revealed. Mitogenomic phylogenetic results indicated that the family Rhabdiasidae is a member of Rhabditoidea in Rhabditina, and is closely related to Rhabditidae. Molecular phylogenies based on the ITS + 28S sequence data supported the validity of Kurilonema, and showed that Kurilonema is sister to Neoentomelas. The present phylogenetic results also indicated that the ancestors of rhabdiasids seem to have initially infected reptiles, then spreading to amphibians. Graphical Abstract Supplementary Information The online version contains supplementary material available at 10.1186/s13071-024-06201-z.

In the present study, to enrich the mitogenomic data and investigate the patterns of mitogenomic evolution of the family Rhabdiasidae, the complete mitochondrial genomes of Rhabdias kafunata Sata, Takeuchi & Nakano, 2020 and R. bufonis (Schrank, 1788) were sequenced and annotated for the first time.Moreover, to assess the evolutionary relationships among the genera within Rhabdiasidae and clarify the systematic position of the Rhabdiasidae in Rhabditida, phylogenetic analyses based on the nuclear large ribosomal subunit (28S) and internal transcribed spacer (ITS) sequences, and the amino acid sequences of 12 proteincoding genes (PCGs) of mitochondrial genomes, were conducted using maximum likelihood (ML) and Bayesian inference (BI).

Parasite collection and species identification
Large numbers of nematodes belonging to the genus Rhabdias were collected from the lung of the Asiatic toad Bufo gargarizans Cantor (Amphibia: Anura) in Shijiazhuang, Hebei Province, China, fixed and stored in 80% ethanol until study.These nematodes were identified to the species level using light and scanning electron microscopy, in addition to the genetic characterization of the nuclear 28S and ITS and mitochondrial cytochrome c oxidase subunit 1 (cox1) and 12S small subunit ribosomal RNA gene.
For light microscopy, nematodes were cleared in glycerin, and then examined and photographed using a Nikon ® optical microscope (Nikon ECLIPSE Ni-U, Nikon corporation, Tokyo, Japan).For scanning electron microscopy (SEM), the anterior and posterior ends of nematodes were transferred to 4% formaldehyde solution, and then post-fixed in 1% O s O 4 , dehydrated via an ethanol series and acetone and critical point dried.The specimens were coated with gold and examined using a Hitachi S-4800 scanning electron microscope (Hitachi Ltd., Tokyo, Japan) at an accelerating voltage of 20 kV.Voucher specimens were deposited in College of Life Sciences, Hebei Normal University, Hebei Province, China.
For molecular procedures, a total of nine nematode specimens (four individuals of R. kafunata and five individuals of R. bufonis) were randomly selected for the following procedures.Genomic DNA from each individual was extracted using a Column Genomic DNA Isolation Kit (Shanghai Sangon, China) according to the manufacturer's instructions.DNA was eluted in elution buffer and kept it at −20 °C until use.The primers and cycling conditions for amplifying different target regions by polymerase chain reaction (PCR) are provided in Additional file 1: Table S1.All PCR reactions were performed in 50 μl consisting of 10 mM Tris HCl at pH 8.4, 50 mM KCl, 3.0 mM MgCl 2 , 250 μM of each dNTP, 50 pmol of each primer, and 1.5 U of Taq polymerase (Takara Bio Inc., Kusatsu, Shiga, Japan) in a thermocycler (model 2720; Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA).
PCR products were checked on GoldView-stained 1.5% agarose gel and purified by the Column PCR Product Purification Kit (Shanghai Sangon, China).Sequencing for each sample was carried out for both strands using a DyeDeoxyTerminator Cycle Sequencing Kit (v.2, Applied Biosystems, California, USA).Sequences of R. kafunata and R. bufonis obtained herein were aligned and compared with genetic data of Rhabdias available in the National Center for Biotechnology Information (NCBI) database (http:// www.ncbi.nlm.nih.gov) using ClustalW2.The 28S, ITS, cox1 and 12S sequences of R. kafunata and R. bufonis were deposited in the Gen-Bank database (http:// www.ncbi.nlm.nih.gov).

Mitochondrial genome sequencing, assembly, and annotation
A total of 30 Gb clean genomic data of each species were generated using the Pair-End 150 sequencing method on the Illumina NovaSeq 6000 platform by Novogene (Tianjin, China).The complete mitochondrial genomes were assembled using GetOrganelle v1.7.2a [24].Protein coding genes (PCGs), ribosomal RNA (rRNAs), and transfer RNA (tRNAs) were annotated using MitoS web server (http:// mitos2.bioinf.uni-leipz ig.de/ index.py) and MitoZ v2.4 [25].The open reading frame (ORF) of each PCG was confirmed manually by the web version of ORF finder (https:// www.ncbi.nlm.nih.gov/ orffi nder/).The "lost" tRNA genes ignored by both MitoS and MitoZ were identified using BLAST on the basis of a database of the existing tRNA sequences of nematodes.The secondary structures of tRNAs were predicted by ViennaRNA module [26], building on MitoS2 [27] and RNAstructure v6.3 [28], followed by manual correction.MitoZ v2.4 was used to visualize and depict gene element features [25].The base composition, amino acid usage, and relative synonymous codon usage (RSCU) were calculated by Python script, which refers to Codon Adaptation Index (CAI) [29].The total length of the base composition included ambiguous bases.The base skew analysis was used to describe the base composition of nucleotide sequences.The complete mitochondrial genomes of R. kafunata and R. bufonis obtained were deposited in GenBank (http:// www.ncbi.nlm.nih.gov).

Phylogenetic analyses
Phylogenetic analyses of rhabdiasid nematodes were performed on the basis of the ITS + 28S sequences using the maximum likelihood (ML) with IQ-TREE [30] and Bayesian inference (BI) with MrBayes [31].Caenorhabditis elegans (Rhabditida: Rhabditidae) was chose as the out-group.The in-group included 47 rhabdiasid species representing six genera belonging to Rhabdiasidae.Detailed information on species included in the phylogenetic analyses is provided in Additional file 2: Table S2.Genes were aligned separately using the MAFFT v7.313 multiple sequence alignment program under the iterative refinement method of E-INS-I [32].In addition, partially ambiguous bases were manually inspected and removed.The aligned and pruned sequences were concatenated into a matrix by PhyloSuite v1.2.2.The TPM2u + F + I + I + R2 model was selected for ML analyses.The GTR + F + G4 models were selected for BI analyses.Reliabilities for ML inference were tested using 1000 BS replications, and BIC analysis was run for 5 × 10 6 MCMC generations.
Phylogenetic analyses were also performed on the basis of concatenated amino acid sequences of 12 PCGs using maximum likelihood (ML) and Bayesian inference (BI).Toxocara cati (Ascaridida: Toxocaridae) was chosen as the out-group in this case.The in-group included 12 species of Tylenchina and 46 species of Rhabditina.Detailed information on the representatives included in this analysis is provided in Additional file 3: Table S3.Substitution models were compared and selected according to the Bayesian Information Criterion by using ModelFinder.For ML inference, the mtZOA + F + R6 model was identified as the optimal nucleotide substitution model.For BI inference, the optimal nucleotide substitution models selected for each partitioning scheme were provided in Additional file 4: Table S4.
In the ML tree, the bootstrap support (BS) values ≥ 80 were considered to constitute strong nodal support, whereas BS values ≥ 50 and < 80 were considered to constitute moderate nodal support.In the BI tree, Bayesian posterior probabilities (BPP) values ≥ 0.98 were considered to constitute strong nodal support, whereas BPP values ≥ 0.95 and < 0.98 were considered to constitute moderate nodal support.BS values ≥ 50 and BPP values ≥ 0.95 are shown in the phylogenetic trees.
Present locality: Shijiazhuang, Hebei Province, China.Site in host: Lung.Prevalence and intensity of infection: 11 of 50 (22.0%)toads examined, and infected with intensity of 1-14 (mean 5.7) nematodes per infected host (a total of 63 female nematode specimens collected).

Morphological description
Small, whitish nematodes, with an conspicuously inflated cuticle along entire body; surface of cuticle has remarkably irregular transverse folds (Figs.1a-f, 2a-c).Body gradually tapers from mid-region toward anterior and posterior ends.Maximum width at region slightly anterior to vulva (Fig. 1a).Lateral alae absent.Lateral cuticular pores distributed laterally in a longitudinal row along entire body (Figs.1b, 3d, e).Cephalic extremity rounded.Oral opening simple, more or less square-shaped, surrounded by six small lips (two lateral and four submedian) reduced to elongated elevations (Figs.2d, 3b); submedian lips located closer to the edge of oral opening than lateral lips, with each lip bearing single papilla (Figs.2d, 3b).Amphids poroid located at base of lateral lip (Figs.2d, 3c).Buccal capsule small and cup-like, with well sclerotized walls (Figs.1c, e, 2a, c).Esophagus clubshaped, with posterior end slightly expanded to esophageal bulb; anterior part of esophagus mostly muscular, posterior part more or less glandular (Figs.1a, c, 2a).Muscular part slightly dilated at its anterior two-thirds.

Molecular characterization of Rhabdias kafunata
Four partial cox1 sequences of R. kafunata obtained here had 655 bp, representing four different genotypes with 0.31-1.22% of nucleotide divergence.Pairwise comparison of the partial cox1 sequences of R. kafunata obtained here with that of R. kafunata (LC496790, LC496791) available in GenBank, showed 2.44% of nucleotide divergence.Three partial 12S sequences of R. kafunata obtained here had 475 bp, with no nucleotide divergence detected.Pairwise comparison of the partial 12S sequences of R. kafunata obtained here with that of R. kafunata (LC496792, LC496793) available in GenBank showed 0.63% of nucleotide divergence.There are no 28S or ITS sequences of R. kafunata available in GenBank.Four partial 28S sequences of R. kafunata obtained here had 553 bp, with no nucleotide divergence detected.Four

Remarks
Sata et al. [33] originally described R. kafunata from B. gargarizans miyakonis in Japan, and also provided the cox1 and 12S sequences for the species.The present specimens collected from B. gargarizans in China are almost identical to the original description of R. kafunata regarding several features, including body length, morphology of buccal capsule, relative length of esophagus to body length, morphology and length of tail, position of vulva, and size of eggs (Table 1 for details).Moreover, molecular analysis showed only 0.63% and 2.44% of nucleotide divergence in the 12S and cox1 sequences, respectively, between the present material and the types of R. kafunata.Therefore, the present specimens were identified as R. kafunata.The detailed morphology of the cephalic structures, vulva, and eggs was observed using SEM for the first time.We also generated 28S and ITS sequences of R. kafunata for the first time, which are useful for the phylogeny and molecular diagnosis of this species.Rhabdias kafunata is then reported in China for the first time, and represents the ninth species of Rhabdias recorded in the country.
Prevalence and intensity of infection: 45 of 50 (90.0%)toads examined, and infected with an intensity of 4-121 (mean 45.0) nematodes per infected host (a total of 1161 female nematode specimens collected).

Molecular characterization of Rhabdias bufonis
Five partial 28S sequences of R. bufonis obtained here had 553-559 bp, with no nucleotide divergence detected.Pairwise comparison of these partial 28S sequences of R. bufonis with that of R. bufonis (KF999593) and R. cf.bufonis (KF999606, KF999609)

Remarks
Rhabdais bufonis has been reported from various frogs and toads worldwide [4,[34][35][36][37][38][39], in which some of these reports are based on redescription of the species [4,34,37].However, some specific identifications of R. bufonis are questionable [4].The present specimens collected from B. gargarizans in China are largely similar to the redescriptions of R. bufonis, regarding several features, including body length, morphology and lengths of esophagus and tail, position of vulva, and size of eggs (Table 2 for details).Therefore, we considered the present material conspecific to R. bufonis.Moreover, genetic sequences showed only 0-0.37%, 0.15%, and 0.86% of nucleotide divergence in the 28S, ITS, and 12S data of R. bufonis available in GenBank.The detailed morphology of the cephalic structures, vulva and eggs of R. bufonis was observed using SEM for the first time.The general morphology of R. bufonis is very similar to that of R. nipponica, but the body length and egg size of R. nipponica are distinctly smaller than that of R. bufonis (body Additionally, R. nipponica has been recorded only infecting frogs and never toads.Marcaida et al. [41] reported the cox1 sequences of R. nipponica collected from different hosts in Japan, which displayed 0.13% (LC671278 versus LC671277) to 9.65% (LC671283 versus LC671275) of nucleotide divergence between different samples.This result seems to suggest that R. nipponica could possibly be a complex comprising several sibling species.The similar situation maybe also occurs in R. bufonis, because molecular analysis of the cox1 and 12S data of R. bufonis between our material and the specimens collected from Rana temporaria in Ukraine [42] showed a low level of nucleotide divergence in 12S region (0.86%), but a high level of nucleotide divergence in the cox1 region (6.24%).Moreover, the specimens of R. bufonis collected from Europe (Germany and Ukraine) showed remarkable morphological variability in body length and relative length of entire esophagus and tail to body length (see Table 2 for details), which may be the result of the different infection intensity, localities, or host species, but may also indicate that the European specimens of R. bufonis possibly represent a species complex.Additionally, the level of nucleotide divergence between R. bufonis and R. engelbrechti are 0.57-1.13% in ITS, 0.56% in 12S, and 4.36-6.63% in the cox1 region, but R. engelbrechti with divided buccal capsule is different from R. bufonis [43].A more rigorous molecular analysis with samples of R. bufonis collected from different localities and hosts worldwide (especially from Europe) is required to solve the taxonomic status of this species.

Phylogenetic analyses
Phylogenetic trees constructed on the basis of the ITS + 28S sequence data using ML and BI had almost Fig. 7 Gene maps of the mitochondrial genomes of Rhabdias kafunata and Rhabdias bufonis.NCR, non-coding region; PCG, protein-coding gene; rRNA, ribosomal RNA; tRNA, transfer RNA identical topologies (Fig. 9).The representatives of Rhabdiasidae were divided into four large clades (clade I, II, III, and IV).Clade I included species of Neoentomelas, Kurilonema, and Serpentirhabdias.Among them, Neoentomelas and Kurilonema clustered together, being sister to Serpentirhabdias.Clade II contained representatives of Entomelas.Clade III consisted of species of Pneumonema.Clade IV included the representatives of Rhabdias.In the clade IV, R. kafunata was sister to R. bulbicauda,, and R. bufonis was sister to R. nipponica.
Phylogenetic results based on the amino acid sequences of 12 PCGs of mitogenomes using ML and BI had almost identical topologies (Fig. 10).The representatives of the Rhabditida were divided into two large monophyletic clades, representing the suborders Tylenchina (clade I) and Rhabditina (clade II), respectively.In clade I (Tylenchina), the superfamily Steinernematoidea (including only Steinernema carpocapsae) was basal.The representative of the superfamily Cephaloboidea (including only Acrobeles complexus) formed a sister lineage to the superfamily Tylenchoidea.
In the clade II, representatives of Rhabditina were divided into four subclades (I, II, III, and IV).In the subclade I (representing the superfamilies Rhabditoidea + Diplogasteroidea), species of the Diplogasteroidea/Diplogasteridae (including Pristionchus pacificus and Allodiplogaster sudhausi) clustered with representatives of Rhabditidae (including Cruznema tripartitum, Caenorhabditis elegans and C. tropicalis), all of which were sister to the family Rhabdiasidae (including  In recent decades, a large number of mitogenomes of nematodes have been published, which contributes to the knowledge regarding their phylogeny and evolutionary history [7,8,44,45].However, several families still have all representatives with unknown mitogenomic data.In the present study, the complete mitochondrial genomes of R. kafunata and R. bufonis were provided for  the first time.The size of the complete mitogenomes of R. kafunata (15,437 bp) and R. bufonis (15,128 bp) were shown to be larger than that of other species belonging to Rhabditina (Dictyocaulus viviparus 13,310 bp to Syngamus trachea 14,647 bp), with exception to that of Mecistocirrus digitatus (15,221 bp).The composition of the complete mitogenomes of R. kafunata and R. bufonis [including 12 PCGs (missing atp8), 22 tRNA genes, and 2 rRNA genes] was typical of most nematode mitogenomes, except for those of Trichinella spp.and Trichuris spp., which have the atp8 gene [46][47][48][49][50].A recent study claimed to sequence the mitogenome of R. kafunata (OP605735) [51].However, this study did not provide any information on the species identification of their specimens.Pairwise comparison of the partial cox1 sequences of R. kafunata obtained here and available in Genbank (LC496790, LC496791) with that of the mitogenome of R. kafunata (OP605735) showed 4.43-4.89% of nucleotide divergence, which is distinctly higher than the level of intraspecific genetic variation of R. kafunata (0.31-2.44%) and is similar to the level of interspecific genetic variation of some Rhabdias spp.(i.e., the level of interspecific genetic variation of R. bufonis and R. engelbrechti is 4.36-6.63% in thecox1 region).Moreover, the size and gene rearrangement of the mitogenome of R. kafunata in Li et al. [51] are also different from the present study.Consequently, we considered the specimens identified as R. kafunata by Li et al. [51] to be not conspecific with R. kafunata.
Kim et al. [52] indicated that mitogenomes of the Enoplea display much higher level of gene rearrangement than that in the members of Chromadorea.Currently, there are 60 types of mitochondrial gene arrangements found in Nematoda, according to the differences in the position of the 12 or 13 PCGs, 22 tRNA genes, and 2 rRNA genes (Fig. 8).In comparison to these types of gene arrangement, one PCG (nad4) and several tRNA genes (tRNA-Ala, tRNA-Met, tRNA-Cys, tRNA-Asp, and tRNA-Gly) rearrangement events occurred in the two rhabdiasid species.Additionally, the present study also revealed the presence of tRNA translocations (tRNA-Met, tRNA-Val/tRNA-Cys), when comparing the mithocondrial genomes of R. kafunata and R. bufonis.Such tRNA translocations are much more common than protein coding gene or rRNA gene translocations in Nematoda, especially in closely related congeneric species [7].However, the mitochondrial gene arrangement in R. kafunata and R. bufonis was consistently different from all of the known mitogenomes of nematodes.
Currently, the phylogenetic knowledge of the family Rhabdiasidae is limited.The present phylogenetic analyses based on ITS + 28S sequences included the most comprehensive taxa sampling of Rhabdiasidae so far.Within this family, the validity of the genus Kurilonema remains under debate.Baker [2] considered Kurilonema to be a synonym of Entomelas.However, such a proposal was rejected in the subsequent studies [4,53,54].The present results supported the validity of Kurilonema, and indicated that this genus is sister to Neoentomelas with strong support in both ML and BI analyses (BS = 100, BPP = 0.999), which are consistent with the morphological hypothesis [53,54].The close affinity between Kurilonema and Neoentomelas can be depicted through the similar shape of buccal capsule, and in the fact that members of these genera are parasites specific to skinks (Scincidae) in eastern Asia [3,54].
The present results supported the monophyly of Entomelas, Pneumonema, Serpentirhabdias, and Rhabdias, but only the branches of Serpentirhabdias spp.and Entomelas spp.have strong support in both ML and BI analyses (BS = 99, BPP = 0.982/0.988).Among them, Pneumonema was sister to Rhabdias, similar to the observations in previous studies [5,[55][56][57][58].In the genus Rhabdias, R. kafunata was closely related to R. bulbicauda with strong support in both ML and BI analyses (BS = 99, BPP = 0.997), but R. bufonis was closely related to R. nipponica with strong support only in ML tree (BS = 95).Baker [59] presumed that the ancestors of the Rhabdiasidae initially parasitized amphibians and colonized reptiles afterwards.However, the present phylogeny indicated that the ancestors of rhabdiasids initially infected reptiles prior to amphibians.
The present study represented the first attempt to resolve the systematic position of Rhabdiasidae using phylogenetic analyses based on mitogenomic data.In this sense, the present results supported the monophyly of Tylenchina and Rhabditina, and indicated that Rhabdiasidae is closely related to Rhabditidae in the superfamily Rhabditoidea of Rhabditina.Therefore, these results are partially consistent with the traditional classification of Anderson & Bain [21], but contrary to other studies [10,23].Some previous studies considered the families Rhabdiasidae and Strongyloididae to have a close relationship [10,21,23], but the present mitogenomic phylogenies rejected this hypothesis and placed Rhabdiasidae and Strongyloididae in two different suborders.Such results are similar to those from previous studies [16,19,20].Moreover, the phylogenetic relationship between Rhabditidae and Diplogasteroidea/Diplogasteridae remains under debate, and some authors suggested that Diplogasteroidea/Diplogasteridae belong to Rhabditidae [12,17,20].The present results supported the allocation of Diplogasteroidea/Diplogasteridae within Rhabditidae/ Rhabditoidea.
A recent classification placed Ancylostomatidae in Strongyloidea, dissolving the superfamily Ancylostomatoidea [23].The present results supported this classification, but the monophyly of the family Ancylostomatidae was not supported, similar to the previous studies [52,60,61].The mitogenomic phylogenies also indicated that the Strongyloidea is closer to Trichostrongyloidea than Metastrongyloidea.

Conclusions
The detailed morphology of R. kafunata and R. bufonis was further studied using light and scanning electron microscopy.The complete mitochondrial genomes of R. kafunata and R. bufonis were reported for the first time.The gene arrangement in the mitogenomes of R. kafunata and R. bufonis represented two new types in Nematoda.The mitogenomic phylogenies indicated that the family Rhabdiasidae is a member of the superfamily Rhabditoidea in Rhabditina, and is closely related to the family Rhabditidae.Molecular phylogenies based on the ITS + 28S sequence data supported the validity of Kurilonema, and showed that Kurilonema is sister to Neoentomelas.The phylogenetic results also indicated that the ancestors of rhabdiasids seem to have infected reptiles first, prior to amphibians.

Fig. 1
Fig. 1 Photomicrographs of Rhabdias kafunata from Bufo gargarizans in China.a Entire body (arrow indicating the vulva), lateral view; b lateral cuticular pores (arrow), lateral view; c anterior part of body, lateral view; d region of vulva, lateral view; e cephalic extremity, lateral view; f posterior part of body (arrow indicating the cloaca), lateral view

Fig. 4
Fig. 4 Photomicrographs of Rhabdias bufonis from Bufo gargarizans in China.a Entire body (arrow indicates vulva), lateral view; b anterior part of body, lateral view; c posterior part of body (arrow indicates cloaca), lateral view; d region of vulva, lateral view; and e lateral cuticular pores (arrow), lateral view

R
. kafunata and R. bufonis).In the subclade II (representing the superfamily Metastrongyloidea), Parafilaroides normani (Filaroididae) nested with the representatives of Metastrongylidae.In the subclade III (representing the superfamily Trichostrongyloidea), the family Heligmonellidae (including only Nippostrongylus brasiliensis) was basal, and the family Molineidae (including Nematodirus oiratianus and N. spathiger) was sister to the representatives of Trichostrongylidae.In the subclade IV (representing the superfamily Strongyloidea), the family Syngamidae (including only Syngamus trachea) was basal.The family Ancylostomatidae (including Necator americanus, Uncinaria sanguinis, Bunostomum phlebotomum, Ancylostoma tubaeforme, and A. caninum) was not monophyletic.Phylogenetic results also indicated the family Strongylidae as a monophyletic lineage.DiscussionIn the present study, 50 individuals of B. gargarizans were examined, with 8 individuals co-infected by R. kafunata and R. bufonis.Rhabdias kafunata can be easily distinguished from R. bufonis by its distinctly longer body and esophagus and much larger buccal capsule (body length 13.3-17.1 mm and esophageal length 0.81-0.90mm and size of buccal capsule 0.020-0.025× 0.028-0.038mm in R. kafunata versus body length 6.10-7.49mm and esophageal length 0.39-0.44mm and size of buccal capsule 0.01-0.013× 0.008-0.010mm in R. bufonis).

Fig. 8 Fig. 9
Fig. 8 Linearized representation of the nematode mitochondrial gene arrangement of nematodes.The non-coding regions are not indicated

Fig. 10
Fig. 10 Maximum likelihood (ML) inference based on the amino acid sequences of 12 protein-coding genes (PCGs) of mitochondrial genomes showing the phylogenetic relationships of representatives of Tylenchina and Rhabditina.Toxocara cati (Ascaridida: Toxocaridae) was chosen as the out-group.Bootstrap values ≥ 50 and Bayesian posterior probabilities values ≥ 0.95 are shown in the phylogenetic trees.Dotted boxes indicated the present phylogenetic results did not supported the validity of the family Diplogasteridae and the superfamily Ancylostomatoidea in the traditional classification

Table 1
Morphometric comparisons of Rhabdias kafunataMeasurements are given in micrometers unless otherwise stated.BL, length of body; BMW, maximum width of body; SBC, size of buccal capsule; EL, length of entire esophagus; AW, width of anterior part of esophagus; DW, width of esophageal dilatation; GW, width of middle glandular part of esophagus; BW, width of posterior end of esophagus; NRC, distance of nerve-ring to cephalic end; EPC, distance of excretory pore to cephalic end; VC, distance of vulva to cephalic end; TL, length of tail; SE, well-developed, embryonated eggs

Table 2
Morphometric comparisons of Rhabdias bufonisMeasurements are given in micrometers unless otherwise stated.BL, length of body; BMW, maximum width of body; SBC, size of buccal capsule; EL, length of entire esophagus; AW, width of anterior part of esophagus; DW, width of esophageal dilatation; GW, width of middle glandular part of esophagus; BW, width of posterior end of esophagus; NRC, distance of nerve-ring to cephalic end; EPC, distance of excretory pore to cephalic end; VC, distance of vulva to cephalic end; TL, length of tail; SE, well-developed, embryonated eggs

Table 3
Base composition and skewness of Rhabdias kafunata and Rhabdias bufonis